Processing and presentation of the rheumatoid arthritis candidate autoantigen aggrecan, by antigen- specific B cells Caroline Louise Wilson A thesis submitted for the degree of Doctor of Philosophy Institute of Cellular Medicine Newcastle University September 2010 Abstract The proteoglycan aggrecan, which is a major structural component of cartilage, has been identified as a candidate autoantigen in rheumatoid arthritis (RA). This is principally due to its degradation early in the disease pathology, its ability to induce an RA-like disease in mouse models and the presence of elevated numbers of reactive T and B cells in RA patients. Studies have also defined an essential requirement for autoantigen-specific B cells as antigen presenting cells (APC) in RA although the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by B cells remains unknown. To investigate the role of autoreactive B cells as APC in RA, I have used two complimentary approaches to generate B cells expressing an aggrecan-specific B cell receptor (BCR). The first was based on a modified monoclonal antibody production protocol and the second involved the transfection of B lymphoma cells with newly generated plasmids encoding an aggrecan-specific BCR. Using the second approach, I have successfully generated aggrecan-specific B cell lines (C71-4C5 and C71-5F10). I have shown that these B cells specifically bind aggrecan leading to efficient processing and the generation of the immunogenic T cell epitope 84-103 that is recognised by both aggrecan-specific T cell hybridomas and CD4+ T cells isolated from an aggrecan-specific TCR transgenic mouse. The aggrecan-specific B cells are able to present aggrecan at least 104 fold more efficiently than non-specific B cells, 102 fold more efficiently than macrophages and comparable to that seen by dendritic cells. By using a panel of inhibitors, I have also shown that the generation of the 84- 103/MHC complex by aggrecan-specific B cells requires an acidic environment, proteolysis by aspartic, serine and metalloproteinases and the “classical” pathway of MHC class II biosynthesis. During this PhD, I have highlighted a novel role for aggrecan-specific B cells as important APC involved in aggrecan-presentation, as well as elucidating a role for metalloproteinases in aggrecan processing and presentation by APC. ii Acknowledgements I would like to thank my supervisor and mentor John for all of his support, encouragement, boundless knowledge and friendship. To Andy for his endless B cell enthusiasm and helpful criticism which has helped me to develop this thesis. To James, Jane and the entire musculoskeletal research group for extending their help, support and advice. I would also like to say a special thanks to Derek and Jelena for their support during the writing of this thesis and for showing me that research really is the best job ever! I must give heartfelt thanks to my fantastic family for supporting and encouraging me throughout all of this PhD thankyou mam, dad, John and Anne. To my boys Andrew and James whom I love with all my heart, who make every day special and who always make me smile no matter what is thrown at me. And finally I would like to dedicate my thesis to Claire and thank her for her unbelievably endless support, patience, love and encouragement. iii Contents Page Abstract ii Acknowledgements iii Contents iv Figures xi Tables xv Abbreviations xvi 1. Introduction 1 1.1 Rheumatoid arthritis. 1 1.2 The synovial joint 5 1.2.1 Articular cartilage 5 1.2.2 Synovium 6 1. 3 The role of T cells in RA 8 1.3.1 Genetic evidence for T cell involvement 8 1.3.2 T cell depletion therapy in RA 10 1.3.3 CD4+ T cell activation and tolerance 11 1.3.4 T cell tolerance 11 1.3.5 The role of Th17/Treg cells in RA 14 1.4 The role of B cells in RA 17 1.4.1 B cell depletion therapy 17 1.4.2 Autoantibodies and their role in disease 18 1.4.3 The role of B cells in lymphoid neogenesis and T cell activation 19 1.4.4 B cells and cytokines 23 1.5 B cells as antigen presenting cells 25 1.5.1 Antigen uptake via BCR 26 1.5.2 The BCR complex 26 1.5.3 Additional B cell antigen receptors 28 1.5.4 B cell Tolerance 29 1.5.5 B cell tolerance breakdown 30 1.6 Autoantigen processing and presentation 32 1.6.1 Candidate autoantigens 32 1.6.2 Antigen uptake by APC 33 iv 1.6.3 MHC class II assembly and trafficking. 35 1.6.4 Antigenic peptide generation and loading of MHC class II molecules 36 1.6.4.1 Proteolysis 36 1.6.4.2 Intracellular pH 39 1.6.4.3 The antigen structure 40 1.6.5 Inhibition of the antigen processing and presentation pathway. 42 1.6.5.1 Cycloheximide and Brefeldin A 43 1.6.5.2 Ammonium chloride 44 1.6.5.3 Proteinase inhibitors 44 1.7 Proteoglycan aggrecan as a candidate joint autoantigen 48 1.7.1 Aggrecan structure and function 48 1.7.2 Aggrecan breakdown and turnover in disease 50 1.7.3 Proteoglycan induced arthritis 53 1.7.4 Humoral and cellular responses to aggrecan in RA patients. 56 1.8 Aims of the project 58 2. Materials and Methods 59 2.1. Antigens 59 2.1.1 Bovine Aggrecan Preparation 59 2.1.1.2 Dissection 59 2.1.1.3 Proteoglycan separation by caesium chloride gradient 59 2.1.1.4 Deglycosylation 60 2.1.1.5 Quantification 60 2.1.1.6 SDS –polyacrylamide gel electrophoresis 60 2.1.1.7 Viscometry 61 2.1.1.8 ELISA 61 2.1.1.9 Biotinylation 62 2.1.1.10 Reduction and alkylation of aggrecan. 62 2.1.2 Recombinant tetanus toxin C fragment (TTCF). 62 2.1.3 Peptides 62 2.2 Cell lines and culture 63 2.3 Cryopreservation of cells. 64 2.3.1 Freezing cells 64 2.3.2 Thawing cells 64 v 2.4 Single cell cloning 64 2.5 Preparation of bone marrow derived dendritic cells for use as APC 65 2.6 Preparation of bone marrow derived macrophages for use as APC 65 2.7 Animals 66 2.8 Immunisations 66 2.9 Generation of immortalized aggrecan-specific B cells by fusion with 67 A20-1 HS lymphoma cells. 2.9.1 Fusion 1 immunisation 67 2.9.2 Fusion 2 immunization 67 2.9.3 Splenocyte/A20-1 HS fusion 67 2.9.4 The use of CD40L transfectants to expand splenic B cells prior to fusion. 68 2.10 Fusion screening 69 2.10.1 Flow cytometry 69 2.10.2 Antigen presentation assay 71 2.10.3 IL-2 bioassay 71 2.11 Antigen presentation assay (fixed APC system) 72 2.11.1 Pre-fixation of APC 72 2.11.2 Post-fixation of APC 72 2.12 Inhibitors of antigen processing 73 2.13 Construction of plasmids encoding aggrecan-specific BCR. 74 2.13.1 Cell lines and plasmids 74 2.13.2 Characterisation of the C71 and 1-C-6 antibodies by ELISA 74 2.13.3 RNA extraction 75 2.13.4 Rapid amplification of cDNA ends 75 2.13.5 Cloning 77 2.13.6 Plasmid DNA isolation 79 2.13.7 DNA sequencing 79 2.13.8 Maxi preps 79 2.13.9 Restriction digests. 80 2.13.10 Gel extraction/purification 81 2.13.11 DNA ligation 81 2.13.12 PCR to introduce restriction sites required for subsequent cloning steps. 81 2.14 Transfection with the aggrecan-specific BCR plasmids 83 vi 2.14.1 Transient transfection of plasmids containing C71/ 1-C-6 BCR 83 into HeLa cells. 2.14.2 Amaxa electroporation 83 2.15 A20-1 HS generation of stable cell transfectants. 84 2.16 Transfectant analysis 85 2.16.1 HeLa transfectant Immunofluorescence 85 2.16.2 A20-1 HS transfectant screening (Flow cytometry). 85 2.16.3 A20-1 HS transfectant screening (Antigen presentation). 86 2.17 Generation of aggrecan-specific T cell hybridomas 87 2.17.1 Immunisation, cell isolation and in vitro re-stimulation. 87 2.16.2 T cell fusion 88 2.17 Cytokine analysis of CD4+ T cells from TCR-5/4E8-tg mice. 89 2.17.1 CD4+ T cell isolation 89 2.17.2 Flow cytometry to determine purity and TCR Vβ4 usage of the 89 isolated CD4+ T cells. 2.17.3 Cytokine analysis 90 2.18 Statistical analysis 91 3. Generation of immortalized aggrecan-specific B cells by fusion with 92 A20-1 HS lymphoma cells. 3.1 Introduction 92 3.1.2 The aims of this chapter 94 3.2 Results 95 3.2.1 Immunisation and cell fusion. 95 3.2.2 Characterisation of fusion cells by flow cytometry and 98 antigen presentation. 3.2.2.1 Flow cytometry 98 3.2.2.2 Antigen presentation assay. 101 3.2.2.3 Detection of aggrecan binding to fusions by flow cytometry. 109 3.2.2.4 Presentation of p84-103 and deglycosylated aggrecan to 112 T cell hybridomas 192 by fusion clones 53.28 and 24.14 in the presence of anti-Ig, anti-class II or anti-Fc to block presentation. 3.2.2.4 Ig characterisation by ELISA. 114 3.2.2.5 Is aggrecan presentation improved due to the extracellular proteinases? 116 vii 3.2.2.6 Aggrecan presentation to T cell hybridomas specific for epitopes 119 within the G3 domain.